A specific radiochemical assay for pyrroline-5-carboxylate dehydrogenase

Previous studies of pyrroline-5-carboxylate dehydrogenase have been conducted using a spectrophotometric method to monitor substrate-dependent NAD(P)H production. For the assay of the mammalian enzyme, the spectrophotometric assay was found to be unacceptable for kinetic studies as the production of NAD(P)H was nonlinear with time and protein concentration. An assay which measures radiolabeled glutamate production by this enzyme in the presence of NAD+ from radiolabeled pyrroline-5-carboxylate has been developed. Separation of substrate from product is achieved by column chromatography using Dowex 50 cation-exchange resin. The product isolated by this procedure was identified as glutamate. This new assay is linear with time and protein concentration and gives reproducible results. The assay is not influenced by competing enzyme activities, such as glutamate dehydrogenase, in a liver homogenate so that quantitative conversion of pyrroline-5-carboxylate to glutamate is observed.